Compounds and compositions with nitrogen-containing non-basic side

ABSTRACT

The present invention provides compounds with nitrogen-containing non-basic side chains of formula II ##STR1## wherein R 1  and R 2 , independently, are H, OH, O(C 1  -C 6  alkyl), O--C(O)--(C 1  -C 6  alkyl), O--C(O)--O(C 1  -C 6  alkyl), O--C(O)--Ar, O--C(O)--O--Ar, O--SO 2  --(C 4  -C 6  alkyl), chloro, fluoro, or bromo; 
     W is CHOH, C(O), or CH 2  ; 
     Y is (CH 2 ) n , or CH(C 1  -C 4  alkyl); 
     V is S, 0, or CH 2  CH 2  ; 
     n is 1, 2, or 3; and 
     Ar is optionally substituted phenyl. 
     The present invention also provides pharmaceutical compositions containing compounds of formula II, optionally containing estrogen or progestin, and the use of such compounds, alone, or in combination with estrogen or progestin, for alleviating the symptoms of post-menopausal symptoms, particularly osteoporosis, cardiovascular related pathological conditions, and estrogen-dependent cancer. 
     The present invention further provides the use of the compounds of the present invention for inhibiting uterine fibroid disease and endometriosis in women and aortal smooth muscle cell proliferation, particularly restenosis, in humans.

FIELD OF THE INVENTION

This invention relates to the fields of pharmaceutical and organicchemistry and provides novel compounds with nitrogen-containingnon-basic side chains, which are useful for the treatment of the variousmedical indications associated with post-menopausal syndrome, anduterine fibroid disease, endometriosis, and aortal smooth muscle cellproliferation. The present invention also relates to pharmaceuticalcompositions of the compounds of the present invention.

BACKGROUND OF THE INVENTION

"Post-menopausal syndrome" is a term used to describe variouspathological conditions which frequently affect women who have enteredinto or completed the physiological metamorphosis known as menopause.Although numerous pathologies are contemplated by the use of this term,three major effects of post-menopausal syndrome are the source of thegreatest long-term medical concern: osteoporosis, cardiovascular effectssuch as hyperlipidemia, and estrogen-dependent cancer, particularlybreast and uterine cancer.

Osteoporosis describes a group of diseases which arise from diverseetiologies, but which are characterized by the net loss of bone mass perunit volume. The consequence of this loss of bone mass and resultingbone fracture is the failure of the skeleton to provide adequatestructural support for the body.

One of the most common types of osteoporosis is that associated withmenopause. Most women lose from about 20% to about 60% of the bone massin the trabecular compartment of the bone within 3 to 6 years after thecessation of mensus. This rapid loss is generally associated with anincrease of bone resorption and formation. However, the resorptive cycleis more dominant and the result is a net loss of bone mass. Osteoporosisis a common and serious disease among post-menopausal women.

There are an estimated 25 million women in the United States, alone, whoare afflicted with this disease. The results of osteoporosis arepersonally harmful and also account for a large economic loss due itschronicity and the need for extensive and long term support(hospitalization and nursing home care) from the disease sequelae. Thisis especially true in more elderly patients. Additionally, althoughosteoporosis is not generally thought of as a life threateningcondition, a 20% to 30% mortality rate is related with hip fractures inelderly women. A large percentage of this mortality rate can be directlyassociated with post-menopausal osteoporosis.

The most vulnerable tissue in the bone to the effects of post-menopausalosteoporosis is the trabecular bone. This tissue is often referred to asspongy or cancellous bone and is particularly concentrated near the endsof the bone (near the joints) and in the vertebrae of the spine. Thetrabecular tissue is characterized by small osteoid structures whichinterconnect with each other, as well as the more solid and densecortical tissue which makes up the outer surface and central shaft ofthe bone. This inter-connected network of trabeculae gives lateralsupport to the outer cortical structure and is critical to thebio-mechanical strength of the overall structure.

In post-menopausal osteoporosis, it is, primarily, the net resorptionand loss of the trabeculae which leads to the failure and fracture ofbone. In light of the loss of the trabeculae in post-menopausal women,it is not surprising that the most common fractures are those associatedwith bones which are highly dependent on trabecular support, e.g., thevertebrae, the neck of the weight bearing bones such as the femur andthe fore-arm. Indeed, hip fracture, collies fractures, and vertebralcrush fractures are hall-marks of post-menopausal osteoporosis.

At this time, the only generally accepted method for treatment ofpost-menopausal osteoporosis is estrogen replacement therapy. Althoughtherapy is generally successful, patient compliance with the therapy islow primarily because estrogen treatment frequently produces undesirableside effects.

Prior to menopause, most women have less incidence of cardiovasculardisease than age-matched men. Following menopause, however, the rate ofcardiovascular disease in women slowly increases to match the rate seenin men. This loss of protection has been linked to the loss of estrogenand, in particular, to the loss of estrogen's ability to regulate thelevels of serum lipids. The nature of estrogen's ability to regulateserum lipids is not well understood, but evidence to date indicates thatestrogen can upregulate the low density lipid (LDL) receptors in theliver to remove excess cholesterol. Additionally, estrogen appears tohave some effect on the biosynthesis of cholesterol, and otherbeneficial effects on cardiovascular health.

It has been reported in the literature that post-menopausal womenundergoing estrogen replacement therapy experience a return of serumlipid concentrations to those of the pre-menopausal state. Thus,estrogen would appear to be a reasonable treatment for this condition.However, the side-effects of estrogen replacement therapy are notacceptable to many women, thus limiting the use of this therapy. Anideal therapy for this condition would be an agent which would regulatethe serum lipid level as does estrogen, but would be devoid of theside-effects and risks associated with estrogen therapy.

The third major pathology associated with post-menopausal syndrome isestrogen-dependent breast cancer and, to a lesser extent,estrogen-dependent cancers of other organs, particularly the uterus.Although such neoplasms are not solely limited to a post-menopausalwomen, they are more prevalent in the older, post-menopausal population.Current chemotherapy of these cancers has relied heavily on the use ofanti-estrogen compounds such as, for example, Tamoxifen. Although suchmixed agonist-antagonists have beneficial effects in the treatment ofthese cancers, and the estrogenic side-effects are tolerable in acutelife-threatening situations, they are not ideal. For example, theseagents may have stimulatory effects on certain cancer cell populationsin the uterus due to their estrogenic (agonist) properties and they may,therefore, be contraproductive in some cases. A better therapy for thetreatment of these cancers would be an agent which is an anti-estrogencompound having negligible or no estrogen agonist properties onreproductive tissues.

In response to the clear need for new pharmaceutical agents which arecapable of alleviating the symptoms of, inter alia, post-menopausalsyndrome, the present invention provides new compounds, pharmaceuticalcompositions thereof, and methods of using such compounds for thetreatment of post-menopausal syndrome and other estrogen-relatedpathological conditions such as those mentioned below. The reduction ofbone density and mass leading to osteoporosis that more rarely occurs inmen is also tied to the loss of hormonal regulation and is, therefore,also a target for therapy according to the compounds and methods of thecurrent invention.

Uterine fibrosis is an old and ever present clinical problem known by avariety of names, including uterine hypertrophy, uterine lieomyomata,myometrial hypertrophy, fibrosis uteri, and fibrotic metritis.Essentially, uterine fibrosis is a condition where there is aninappropriate deposition of fibroid tissue on the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibrosis involves surgicalprocedures both costly and sometimes a source of complications such asthe formation of abdominal adhesions and infections. In some patients,initial surgery is only a temporary treatment and the fibroids regrow.In those cases a hysterectomy is performed which effectively ends thefibroids but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, yettheir use is tempered by the fact they can lead to osteoporosis.

Endometriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release and subsequent ovarian production ofestrogen; however, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundersirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinswhich induces amenorrhea and by suppressing ovarian estrogen productioncan cause regressions of the endometrial growths. The use of chronicprogestin therapy is often accompanied by the unpleasant CNS sideeffects of progestins and often leads to infertility due to suppressionof ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis; however, theyinduce severe masculinizing effects. Several of these treatments forendometriosis have also been implicated in causing a mild degree of boneloss with continued therapy. Therefore, new methods of treatingendometriosis are desirable.

Aortal smooth muscle cell proliferation plays an important role indiseases such as atherosclerosis and restenosis. Vascular restenosisafter percutaneous transluminal coronary angioplasty (PTCA) has beenshown to be a tissue response characterized by an early and late phase.The early phase occuring hours to days after PTCA is due to thrombosiswith some vasospasms while the late phase appears to be dominated byexcessive proliferation and migration of aortal smooth muscle cells. Inthis disease, the increased cell motility and colonization by suchmuscle cells and macrophages contribute significantly to thepathogenesis of the disease. The excessive proliferation and migrationof vascular aortal smooth muscle cells may be the primary mechanism tothe reocclusion of coronary arteries following PTCA, atherectomy, laserangioplasty and arterial bypass graft surgery. See "IntimalProliferation of Smooth Muscle Cells as an Explanation for RecurrentCoronary Artery Stenosis after Percutaneous Transluminal CoronaryAngioplasty," Austin et al., Journal of the American College ofCardiology 8:369-375 (Aug. 1985).

Vascular restenosis remains a major long term complication followingsurgical intervention of blocked arteries by percutaneous transluminalcoronary angioplasty (PTCA), atherectomy, laser angioplasty and arterialbypass graft surgery. In about 35% of the patients who undergo PTCA,reocclusion occurs within three to six months after the procedure. Thecurrent strategies for treating vascular restenosis include mechanicalintervention by devices such as stents or pharmacologic therapiesincluding heparin, low molecular weight heparin, coumarin, aspirin, fishoil, calcium antagonist, steroids, and prostacyclin. These strategieshave failed to curb the reocclusion rate and have been ineffective forthe treatment and prevention of vascular restenosis. See "Prevention ofRestenosis after Percutaneous Transluminal Coronary Angioplasty: TheSearch for a `Magic Bullet`", Hermans et al., American Heart Journal122: 171-187 (July 1991).

In the pathogenesis of restenosis, excessive cell proliferation andmigration occurs as a result of growth factors produced by cellularconstituents in the blood and the damaged arterial vessel wall, whichfactors mediate the proliferation of smooth muscle cells in vascularrestenosis.

Agents that inhibit the proliferation and/or migration of aortal smoothmuscle cells are useful in the treatment and prevention of restenosis.The present invention provides for the use of compounds as aortal smoothmuscle cell proliferation inhibitors and, thus, inhibitors ofrestenosis.

SUMMARY OF THE INVENTION

The present invention provides compounds with nitrogen-containingnon-basic side chains of formula I ##STR2## wherein R¹ and R²,independently, are H, OH, O(C₁ -C₆ alkyl), O--C(O)--(C₁ -C₆ alkyl),O--C(O) --O(C₁ -C₆ alkyl), O--C(O)--Ar, O--C(O)--O--Ar, O--SO₂ --(C₄ -C₆alkyl), chloro, fluoro, or bromo;

V is S, 0, or CH₂ CH₂ ;

W is CHOH, C(O), or CH₂ ;

X is (CH₂)n, or (CH₂)_(m) C(O);

R³ and R⁴ each, independently, are H, C₁ -C₆ alkyl, C(O)--(C₁ -C₆alkyl), C(O)--NH--(C₁ -C₆ alkyl), C(O)--Ar, or together with thenitrogen to which they are attached form 1-pyrrolidinyl, 1-piperidinyl,or a 5- or 6-membered imide or cyclic amide;

m is 1 or 2;

n is 1, 2, or 3; and

Ar is optionally substituted phenyl;

provided that at least one of X, R³, and R⁴ contain a carbonylfunctional group.

The present invention also provides compounds with nitrogen-containingnon-basic side chains of formula II ##STR3## wherein R¹ and R²,independently, are H, OH, O(C₁ -C₆ alkyl), O--C(O)--(C₁ -C₆ alkyl),O--C(O)--O(C₁ -C₆ alkyl), O--C(O)--Ar, O--C(O)--O--Ar, O--SO₂ --(C₄ -C₆alkyl), chloro, fluoro, or bromo;

V is S, O, or CH₂ CH₂ ;

W is CHOH, C( 0 ), or CH₂ ;

Y is (CH₂ ) n, CH (C₁ -C₄ alkyl);

n is 1, 2, or 3; and

Ar is optionally substituted phenyl.

The present invention further provides compounds withnitrogen-containing non-basic side chains of formula III ##STR4##wherein R¹ and R², independently, are H, OH, O(C₁ -C₆ alkyl),O--C(O)--(C₁ -C₆ alkyl), O--C(O)--O(C₁ -C₆ alkyl), O--C(O)--Ar,O--C(O)--O--Ar, O--SO₂ --(C₄ -C₆ alkyl), chloro, fluoro, or bromo;

V is S, 0, or CH₂ CH₂ ;

W is CHOH, C(O), or CH₂ ;

Z is a bond or CH₂ ;

R⁵ is C(O)--(C₁ -C₆ alkyl); and

Ar is optionally substituted phenyl.

Compounds of the current invention may have an asymmetric center. Thus,such compounds can have an R- or S-configuration, or a mixture thereof.All such isomers are considered part of this invention.

The present invention also provides pharmaceutical compositionscontaining compounds of formula I, formula II, and formula III,optionally containing estrogen or progestin, and the use of suchcompounds, alone, or in combination with estrogen or progestin, foralleviating the symptoms of post-menopausal symptoms, particularlyosteoporosis, cardiovascular related pathological conditions, andestrogen-dependent cancer. As used herein, the term "estrogen" includessteroidal compounds having estrogenic activity such as, for example,17β-estradiol, estrone, conjugated estrogen (e.g., Premarin®), equineestrogen, 17α-ethynyl estradiol, and the like. As used herein, the term"progestin" includes compounds having progestational activity such as,for example, progesterone, norethynodrel, norgestrel, megestrol acetate,norethindrone, and the like.

The present invention further provides the use of the compounds of thepresent invention for inhibiting uterine fibroid disease andendometriosis in women and aortal smooth muscle cell proliferation,particularly restenosis, in humans.

DETAILED DESCRIPTION OF THE INVENTION

General terms used in the description of compounds of the presentinvention bear their usual meanings. For example, "C₁ -C₄ alkyl" refersto aliphatic chains of 1 to 4 carbon atoms including methyl, ethyl,propyl, isopropyl, butyl, n-butyl, and the like; and "C₁ -C₆ alkyl"encompasses the groups included in the definition of "C₁ -C₄ alkyl" inaddition to groups such as pentyl, isopentyl, hexyl, isohexyl, and thelike. "C₄ -C₆ alkyl" refers to aliphatic chains of 4 to 6 carbon atomsincluding butyl, n-butyl, pentyl, isopentyl, hexyl, isohexyl, and thelike.

The term "substituted phenyl" refers to a phenyl group having one ormore substituents selected from the group consisting of C₁ -C₄ alkyl, C₁-C₅ alkoxy, hydroxy, nitro, chloro, fluoro, or tri(chloro orfluoro)methyl. "C₁ -C₅ alkoxy" represents a C₁ -C₅ alkyl group attachedthrough an oxygen bridge such as, for example, methoxy, ethoxy,n-propoxy, isopropoxy, and the like.

It should also be understood that as used herein, references to alkyland alkoxy structures also include cycloalkyl and cycloalkoxy groupswhere the number of carbons within the structure is at least 3.

Further, "imide" is understood to indicate a heterocyclic structurewherein a nitrogen atom is adjacent to two carbonyl functional groups.An "amide" is understood to be a structure having a nitrogen atomadjacent to a single carbonyl functional group, such amide may becyclic.

Preferred compounds of this invention include compounds of formula Iwherein any or all of the following limitations apply: V is S; W isC(O); and x is (CH₂)₂ or CH₂ C(O), especially (CH₂)₂. Especiallypreferred compounds of formula I are those wherein all of the precedinglimitations apply.

Other preferred compounds of formula I include those compounds whereinR¹ and R² are OH, O--C(O)--(C₁ -C₆ alkyl), O--C(O)--O(C₁ -C₆ alkyl),O--C(O)--Ar, or O--C(O)--O--Ar, especially OH or OCH₃. Of these,compounds wherein R¹ and R² are the same as one another are particularlypreferred.

Certain R³ and R⁴ groups also demonstrate preferable characteristics.For example, those compounds of formula I wherein R³ and R⁴ togetherwith the nitrogen to which they are attached form 1-pyrrolidinyl,1-piperidinyl, or a 5- or 6-membered imide or cyclic amide arepreferred. A further preferred subgroup of the preferred 1-pyrrolidinyl,1piperidinyl, imide, and cyclic amide compounds include those compoundswherein R¹ and R² are OH or OCH₃.

Most especially preferred compounds of formula I include those havingall of the aforementioned limitations, that is, compounds wherein V isS; W is C(O); X is (CH₂)₂ or CH₂ C(O), especially (CH₂)₂ ; R¹ and R² areOH, O--C(O)--(C₁ -C₆ alkyl), O--C(O)--O(C₁ -C₆ alkyl), O--C(O)--Ar, andO--C(O)--O--Ar, especially OH or OCH₃, particularly wherein R¹ and R²are the same as one another; and R³ and R⁴, together with the nitrogento which they are attached form 1-pyrrolidinyl, 1-piperidinyl, or a 5-or 6-membered imide or cyclic amide.

In keeping with the scope of this invention, the preferred compounds offormula I are limited to those wherein at least one carbonyl functionalgroup is present at a position adjacent to the nitrogen in the 3- sidechain. That is, at least one of X, R³, and R⁴ must contain a carbonylfunctional group.

Other preferred compounds of this invention include compounds of formulaII wherein any or all of the following limitations apply: V is S; W isC(O); and Y is CH₂ or CH(CH₃). Especially preferred compounds of formulaII are those wherein all of the preceding limitations apply.

Other preferred compounds of formula II include those compounds whereinR¹ and R² are OH, O--C(O)--(C₁ -C₆ alkyl), O--C(O)--O(C₁ -C₆ alkyl),O--C(O)--Ar, or O--C(O)--O--Ar, especially OH or OCH₃. Of these,compounds wherein R¹ and R² are the same as one another are particularlypreferred.

Yet other preferred compounds of this invention include compounds offormula III wherein any or all of the following limitations apply: V isS; W is C(O); and Z is a bond or CH₂. Especially preferred compounds offormula III are those wherein all of the preceding limitations apply.

Other preferred compounds of formula II include those compounds whereinR¹ and R² are OH, O--C(O)--(C₁ -C₆ alkyl), O--C(O)--O(C₁ -C₆ alkyl),O--C(O)--Ar, or O--C(O)--O--Ar, especially OH or OCH₃. Of these,compounds wherein R¹ and R² are the same as one another are particularlypreferred.

Preferred methods of this invention obviously include those whereinpreferred compounds are used.

The compounds of the present invention are derivatives ofbenzo[b]thiophene which is named and numbered according to the RingIndex, The American Chemical Society, as follows. ##STR5##

In the processes for preparing the compounds of the present invention,the starting material is generally a precursor of formula below, whichcan be prepared via known procedures. ##STR6##

Typically, the two hydroxy groups are protected by known hydroxyprotecting groups that are capable of resisting acylation under standardFriedel-Crafts conditions and subsequent reduction by a strong reducingagent. Preferred hydroxy protecting groups are C₁ -C₄ alkyl, and methylis especially preferred. See, e.g., U.S. Pat. Nos. 4,133,814; 4,380,635;and 4,418,068, each of which is herein incorporated by reference, J. W.Barton, "Protective Groups in Organic Chemistry", J. G. W. McOmie (ed.),Plenum Press, New York, N.Y., 1973, Chapter 2, and T. W. Green,"Protective Groups in Organic Synthesis", John Wiley and Sons, New York,N.Y., 1981, Chapter 7.

Following preparation of the desired protected precursor, the precursoris acylated, using standard FriedelCrafts conditions, according toacylation methods disclosed in the above-incorporated United Statespatents.

All reagents obtained from commercial sources were used without furtherpurification unless otherwise indicated. ¹ H-NMR and ¹³ C-NMR weremeasured as indicated at 300 and 75MHz respectively. ¹ H-NMR chemicalshifts are reported as δ values in ppm relative to the NMR solventemployed. ¹ H-NMR coupling constants are reported in Hertz (Hz) andrefer to apparent multiplicities. Multiplicity is indicated as follows:s (singlet), d (doublet), t (triplet), q (quartet), m (multipier), comp(complex), br (broad), and app (apparent). Column chromatography wasperformed according to the method of Still et. al. (Still, W. C.; Kahn,M.; Mitra, A. J. Org. Chem. 1978, 2923) unless otherwise indicated withEM Science silica gel (230-400 mesh ASTM). Radial chromatography wasperformed on a Chromatotron (Harrison Research) using 1, 2, or 4 mmthick plates. All air and/or moisture sensitive reactions were run underan argon or nitrogen atmosphere in rigorously dried glassware. In allcases, concentrations were performed under reduced pressure with arotary evaporator.

Four general synthetic routes, which were used to prepare compounds ofthe present invention, are outlined below, wherein R¹ and R² are asdefined above. ##STR7##

Compounds of the present invention in which W is CHOH are preparedfollowing sodium ethanethioate deprotection by dissolution in anappropriate solvent and reaction with reducing agent, such as, forexample, lithium aluminum hydride, under an inert gas such as nitrogen.

The amount of reducing agent used in this reaction is an amountsufficient to reduce the carbonyl group to an alcoholic group (CHOH).Generally, an excess of the reducing agent per equivalent of thesubstrate is used.

Suitable solvents include any solvent or mixture of solvents that willremain inert Under reducing conditions, such as, for example, diethylether, dioxane, and tetrahydrofuran (THF). The anydrous form of thesesolvents is preferred, and anhydrous THF is especially preferred.

The temperature employed in this step is that which is sufficient toeffect completion of the reduction reaction. Ambient temperature, in therange from about 17° C. to about 25° C., generally is adequate.

The length of time for this step is that amount necessary for thereaction to occur. Typically, this reaction takes from about 1 to about20 hours. The optimal time can be determined by monitoring the progressof the reaction via conventional chromatographic techniques.

A compound of the present invention wherein W is CHOH may be furtherreduced to provide compounds wherein W is methylene via standardprocedures. This is accomplished by suspending the compound in anappropriate solvent and cooling under an inert gas such as nitrogen. Tothis suspension is added a suitable trialkyl silane reducing agent,preferrably triethyl silyl, and a reasonably strong protic acid such ashydrochloric acid, trifluoroacetic acid, and the like.

Suitable solvents can be any solvent or mixture of solvents that remaininert under the reaction conditions employed in the process. Forexample, halogenated alkane solvents such as dichloromethane and1,2-dichloroethane, as well as haloaromatics such as chlorobenzene andthe like may be used. Of these, dichloromethane is preferred.

The temperature employed in this step is that which is sufficient toeffect completion of the present reduction process. Typically, thereaction is cooled to about 0° C. and the reaction solution is kept onice until the reaction is complete; however, ambient temperature also issatisfactory. In general, this reaction is completed in less than threehours, and the progress of the reaction can be monitored via standardtechniques. The product of this reaction is extracted and purified viastandard techniques.

Alternatively, ketones of the type shown in general route #1 prior toalkylation can be reduced to the compound wherein W is methylene. Inthis process, the R¹ and R² hydroxy protecting groups, which arepreferrably methyl, optionally are removed, and the protected ordeprotected compound is reacted with a reducing agent such as lithiumaluminum hydride in the presence of an inert solvent having a boilingpoint in the range from about 150° C. to about 200° C. While each stepof this process is preferrably carried out in separate vessels, it ispossible to carry out each step of the present process in the samevessel.

The amount of reducing agent used in this reaction is an amountsufficient to reduce the carbonyl group to a methylene group. Generally,an excess of the reducing agent per equivalent of the substrate is used.

The solvent used in the present process is required to have a relativelyhigh boiling point, in the range from about 150° C. to about 200° C., asrepresented by solvents such as, for example, n-propylbenzene, diglyme(1,1'-oxybis[2-methoxyethane]), and anisole, and Red-A1® (sodiumbis(2-methoxyethoxyaluminum hydride)), which also is used as thereducing agent. When the R¹ and R² substituents of compounds of thepresent invention are hydroxy protecting groups, n-propylbenzene is thepreferred solvent. When such protecting groups are first optionallyremoved prior to reduction, Red-A1 is the preferred reagent.

The temperature used in this reaction is that which is sufficient tocomplete the reduction reaction. Preferrably, the reaction mixture isheated to reflux for about 15 minutes to about 6 hours, and allowed tocool to ambient temperature. When R¹ and R² are hydroxy protectinggroups, a small amount of deionized water is added to the mixturefollowed by the addition of a small aliquot of 15% sodium hydroxide.When R¹ and R² are OH, the reaction is carefully quenched with excess1.0N hydrochloric acid. The optimal amount of time for these reactionsto run, typically from about 10 minutes to about 3 hours, can bedetermined by monitoring the progress of the reaction via standardtechniques.

Following reduction of W to CHOH or CH₂, the appropriate groups can beappended on as described previously.

When a O--C(O)--(C₁ -C₆ alkyl) or O--C(O)--Ar group is desired at R¹ andR², a dihydroxy compound of formula I, II, or III is reacted with anagent such as acyl chloride, bromide, cyanide, or azide, or with anappropriate anhydride or mixed anhydride. The reactions are convenientlycarried out in a basic solvent such as pyridine, lutidine, quinoline orisoquinoline, or in a tertiary amine solvent such as triethylamine,tributylamine, methylpiperidine, and the like. The reaction also may becarried out in an inert solvent such as ethyl acetate,dimethylformamide, dimethylsulfoxide, dioxane, dimethoxyethane,acetonitrile, acetone, methyl ethyl ketone, and the like, to which atleast one equivalent of an acid scavenger, such as a tertiary amine, hasbeen added. If desired, acylation catalysts such as4-dimethylaminopyridine or 4-pyrollidinopyridine may be used. See.,e.g., Haslam, et al., Tetrahedron, 36:2409-2433 1980).

The acylation reactions which provide the aforementioned R¹ and R²groups are carried out at moderate temperatures in the range from about-25° C. to about 100° C., frequently under an inert atmosphere such asnitrogen gas. However, ambient temperature is usually adequate for thereaction to run.

Such acylations of the hydroxy group also may be performed byacid-catalyzed reactions of the appropriate carboxylic acids in inertorganic solvents or heat. Acid catalysts such as sulfuric acid,polyphosphoric acid, methanesulfonic acid, and the like are used.

The aforementioned R¹ and R² groups also may be provided by forming anactive ester of the appropriate acid, such as the esters formed by suchknown reagents as dicyclohexylcarbodiimide, acylimidazoles,nitrophenols, pentachlorophenol, N-hydroxysuccinimide, and1-hydroxybenzotriazole. See, e.g., Bull. Chem. Soc. Japan, 38.:1979(1965), and Chem, Ber., 788 and 2024 (1970).

Each of the above techniques that provide O--C(O)--(C₁ -C₆ alkyl) andO--C(O)--Ar groups are carried out in solvents as discussed above. Thesetechniques, which do not produce an acid product in the course of thereaction, of course, do not necessitate the use of an acid scavenger inthe reaction mixture.

When a compound is desired in which R¹ and R² is O--SO₂ --(C₄ -C₆alkyl), a dihydroxy compound is reacted with, for example, a derivativeof the appropriate sulfonic acid such as a sulfonyl chloride, bromide,or sulfonyl ammonium salt, as taught by King and Monoir, J. Am. Chem.Soc., 9.7.:2566-2567 (1975). The dihydroxy compound also can be reactedwith the appropriate sulfonic anhydride. Such reactions are carried outunder conditions such as were explained above in the discussion ofreaction with acid halides and the like.

Compounds of formula I, II, and III can be prepared so that R¹ and R²are different biological protecting groups or, preferably, the samebiological protecting group. Preferred protecting groups include OCH₃,O--C(O)--C(CH₃)₃, O--C(O)--C₆ H5, and O--SO₂ --(CH₂)₃ --CH₃.

The term "biological protecting groups" refers to those R¹ and R²substituents which delay, resist, or prohibit removal of such groups ina biological system such as, for example, following administration of acompound of the present invention containing the above-described R¹ andR² groups to a human. Such compounds also are useful for the methodsherein described, especially when W is CH₂.

The following preparations and examples are presented to furtherillustrate the preparation and use of compounds of the presentinvention. It is not intended that the invention be limited in scope byreason of any of the following preparations and examples. The compoundnumbers correspond to those given in Table 1.

Preparation 1

Preparation of compound 1: ##STR8##

p-Anisoyl chloride (1.54 g, 9.00 mmol, Aldrich Chemical Company) wasdissolved in anhydrous CH₂ C₁₂ (100 ml). To this stirred solution wasadded 6-methoxyphenyl-2-(4-methoxyphenyl)-benzo[b]thiophene (1.62 g,6.00 mmol) prepared by the method of Jones et al. (J. Med. Chem. 1984,27:1057). The resulting mixture was cooled to 0° C., and AlCl₃ (1.20 g,9.00 mmol) was added in small portions over a five minute period. Afterone hour the reaction mixture was poured into ice water (150 ml) andextracted with CH₂ Cl₂ (3×75 ml). The organic layers were combined andwashed with 1N NaOH (30 ml), water (25 ml), and brine (25 ml). Theorganic layers were then dried over MgSO₄. After removal of the solvent,the resulting crude product was flash chromatographed on a silica gelcolumn (eluent: ethyl acetate:hexanes; 3:7) giving 2.253 g (93%) of alight yellow solid. The product was further purified byrecrystallization from acetone/methanol to yield 2.109 g (87%) ofcompound 1.

IR (CHCl₃)ν_(max) 3020, 3015, 2970, 2940, 2840, 1600, 1475, 1253, 1218,1167; ¹ H-NMR (300 MHz, DMSO d₆) δ7.64-7.69 (m, 3H), 7.29-7.32 (m, 3H).6.86-7.00 (m, 5H), 3.83 (s, 3H) 3.76 (s, 3H). ¹³ C-NMR (75.489 MHz, DMSOd₆) δ192, 163.61, 159.47, 157.35, 141, 139.36, 133.17,131.81, 130,129.63,125.17, 123.26, 115.00, 114.35, 114.07, 105.11, 55.49, 55.13; FD⁺-MS for C₂₄ H₂₀ O₄ S=404. Elemental Analysis C₂₄ H₂₀ O₄ S - Calculated:C, 71.27; H, 4.98; S, 7.93; O, 15.82; Found: C, 71.50; H, 5.00; S, 7.98;0, 15.77.

Preparation 2

Preparation of compound 2: ##STR9##

Compound 1 (0.405 g, 1.00 mmol) was dissolved in 2 ml of dry DMF. Tothis stirred solution was added 3.0 ml of 0.50M sodium ethanethioate(NaEtS) in DMF. The reaction temperature was increased to 80° C. forfour hours. The reaction was diluted with ethyl acetate (10 ml), andwater was added (10 ml). The mixture was then neutralized with 1N HC₁and extracted with ethyl acetate (3×20 ml). The organic extracts werecombined, washed with brine (4×20 ml), dried over MgSO4, and evaporatedunder reduced pressure to give a pale yellow solid. The solid wasfurther purified by radial chromatography (2 mm plate, eluting solvent5% ethyl acetate/CH₂ Cl₂). Yield of compound 2 as a foamy yellow solidwas 0.307 g (79%).

IR (CHCl₃)ν_(max) 3585, 3265, 3022, 3012, 2970, 2940, 2840, 1602, 1476,1254, 1163; ¹ H-NMR (CDCl₃) 67 7.70-7.73 (d,2H, J=8.6 Hz), 7.52-7.55 (d,1H, J=8.5 Hz), 7.31-7.34 (m,3H), 6.94-6.98 (dd, 1H, J=9.0 Hz, J=2.4 Hz),6.73-6.76 (d, 2H, J=S.7 Hz), 6.66-6.69 (d, 2H, J=9.1 Hz), 3.88 (s, 3H),3.74 (s, 3H); ¹³ C-NMR (CDCl₃) δ192.92, 159.95, 158.58, 156.47,141.91,138.89, 132.71, 131.67, 129.16, 129.09, 128.85, 124.72, 122.82, 114.27,113.70, 112.95, 103.39, 54.49, 54.08; FD⁺ -MS for C₂₃ H1804S =390.Elemental Analysis C₂₃ H₁₈ O₄ S - Calculated: C, 70.75; H, 4.65; Found:C, 70.93; H, 4.56.

EXAMPLE 1

Preparation of compound 25: ##STR10##

Compound 2 (1.23 g, 3.17 mmol) and 1-trimethylacetylpiperidine-2-methanol (1.58 g, 7.91 mmol) were dissolved in anhydrousTHF (50 ml). To this stirred solution was added PPh₃ (1.66 g, 6.33 mmol)followed by diethylazodicarboxylate (DEAD) (1.00 ml, 6.33 mmol) viasyringe, and the reaction mixture was stirred at room temperature for 18hours. The solvent was removed under reduced pressure, and the resultingmixture was rotary chromatographed (dichloromethane eluent) to yield1.64 g (91%) of compound 25 as product.

IR (CHCl₃)ν_(max) 3008, 2943, 1615, 1601, 1476, 1254, 1165; ¹ H-NMR(CDCl₃) 67.74-7.77 (d, 2H, J=9 Hz), 7.48-7.51 (d, 1H, J=9 Hz), 7.31-7.35(m, 3H), 6.93-6.96 (dd, 1H, J=9 Hz, J=2 Hz), 6.74-6.80 (m, 4H), 3.88 (s,3H), 3.75 (s, 3H), 1.3-4.4 (m, 20H); ¹³ C-NMR (CDCl₃)ν193.26, 177.26,162.87, 159.75, 157.65, 142.42, 140.07, 133.99, 132.36, 130.58, 130.25,126.01, 124.03, 114.79, 114.24, 114.09, 104.51, 65.84, 61.75, 55.63,55.25, 38.94, 28.46, 27.19, 25.49, 25.04, 19.35; FD⁺ -MS for C₃₄ H_(37O)₅ S=571.

EXAMPLE 2

Preparation of compound 22: ##STR11##

Compound 25 (1.24 g, 2.17 mmol) was dissolved in CH₂ Cl₂ (50 ml). Tothis stirred solution was added ethanethiol (EtSH) (0.80 ml, 10.8 mmol)and AlCl₃ (1.73 g, 13.0 mmol). This reaction mixture was stirredvigorously for 30 minutes, and then quenched with brine and saturatedNaHCO₃. Any residue was dissolved by the addition of methanol and ethylacetate. The pH was adjusted to just basic. The mixture was then dilutedwith ethyl acetate (200 ml). After separation of the aqueous layer, theorganic layer was washed with potassium sodium tartrate (3×75 ml) andthen brine (2×75 ml). The organic ethyl acetate layer was dried overMgSO₄ and evaporated under reduced pressure. The product was isolated byrotary chromatography (4 mm plate, eluting solvent 5:4:1 ethylacetate:hexanes:triethylamine) yielding 0,929 g of compound 22 as ayellow solid (79%).

IR (CHCl₃)V_(max) 3298, 3025, 3010, 2946, 1600, 1262, 1166; ¹ H-NMR(MeOD d₄) δ7.68-7.71 (d, 2H, J=8.8 Hz), 7.38-7.41 (d, 1H, J=8.7 Hz),7.24-7.25 (d, 1H, J=2.5 Hz), 7.16-7.19 (d, 2H, J=8.6 Hz), 6.83-6.87 (m,3H), 6.60-6.63 (d, 2H, J=8.6 Hz), 1.35-4.20 (m, 11H), 1.25 (s, 9H); ¹³C-NMR (MeOD d₄) δ194.39, 178.33,163.40, 158.07, 155.62, 142.72,140.28,133.19, 132.37, 130.62, 130.24, 130.09, 124.89, 123.62, 115.38, 114.93,114.28, 106.82, 65.17, 50.36, 38.89, 27.70, 25.53, 25.16, 19.09; FD⁺ -MSfor C₃₂ H₃₃ O₅ S=543; Elemental Analysis C₃₂ H₃₃ O₅ S - Calculated: C,70.69; H, 6.12; N, 2.58; Found: C, 70.47; H, 6.13; N, 2.34.

EXAMPLE 3

Preparation of compound 11: ##STR12##

Compound 2 (3.0 g, 7.69 mmol) was dissolved in DMF (100 ml) and heatedto 70° C. To this stirred solution was added K₂ CO₃ (10.6 g, 76.8 mmol)followed by N,N-Dimethyl chloroacetamide (3.74 g, 30.8 mmol). Thereaction mixture was heated to 100° C. and allowed to stir for 24 hours.The solvent was removed under reduced pressure, and the resultingmixture was dissolved in MeOH and salts were filtered. The crudereaction mixture was recrystallized in H₂ O/methanol (2:1) to yield 2.81g (77%) of compound 11 as product.

¹ H-NMR (CDCl₃) δ7.90 (d, 2H, J=9.7 Hz), 7.65 (d, 1H, J=9.7 Hz), 7.45(d, 2H, J=9.7 Hz), 7.43 (s, 1H), 7.08 (dd, 1H, J=9.7 Hz), 6.93 (d, 2H,J=9.7 Hz), 6.87 (d, 2H, J=9.7 Hz), 4.78 (s, 2H), 4.00 (s, 3H), 3.87(s,3H), 3.16(s, 3H), 3.08(s, 3H); FD⁺ -MS=475; Elemental Analysis C₂₇ H₂₅NO₅ S - Calculated: C, 68.19; H, 5.30; N, 2.94; Found: C, 68.46; H,5.18; N, 2.99.

Preparation 3

Preparation of compound 27: ##STR13##

To a solution of phenol, compound 2 (5.0 g, 12.8 mmol) stirring in DMFat room temperature was added K₂ CO₃ (5.3 g, 38.4 mmol) followed bymethyl bromoacetate (8 ml, 84.5 mmol). The solution was heated to 80° C.for 1 h then cooled to room temperature and poured into brine/ethylacetate (300 ml, 1:1). The mixture was extracted with ethyl acetate(3×100 ml) and the combined organic extracts washed thoroughly withbrine, dried (MgSO₄) and filtered. Concentration gave a yellow syrupwhich was further dried under reduced pressure to yield 5.33 g (90%) of27 as a white crystalline solid which was used without furtherpurification.

¹ H-NMR (CDCl₃) δ7.78 (d, J=8.9 Hz, 2H), 7.51 (d, J=8.5 Hz, 1H),7.30-7.35 (m, 3H), 6.96 (dd, J=9.0, 2.3 Hz, 1H), 6.72-6.78 (overlappingd, 4H), 4.62 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 3.74 (s, 3H).

EXAMPLE 4

Preparation of compound 7: ##STR14##

Reaction of compound 27 (0.42 g, 0.91 mmol), piperidinehydrochloride(0.56mg, 4.58 mmol), and Al(CH₃)₃ (2.29 ml, 4.58 mmol) yielded 0.42 g(90%) of compound 7 as a tan foam.

¹ H-NMR (CDCl₃) δ7.79 (d, J=9.0 Hz, 2H), 7.52 (d, J=8.8 Hz, 1H),7.30-7.35 (m, 3H), 6.96 (dd, J=9.0 Hz, 2.9 Hz, 1H), 6.82 (d, J=8.7 Hz,2H), 6.78 (d, J=8.9 Hz, 2H), 4.66 (s, 2H), 3.90 (s, 3H), 3.75 (s, 3H),3.53 (t, J=4.2 Hz, 2H), 3.42 (t, J=4.2 Hz, 2H), 1.49-1.69 (series of m,6H); IR (CHCl₃) 1639 cm⁻¹ ; FD⁺ -MS 515 (M⁺).

Preparation 4

Preparation of compound 28: ##STR15##

To compound 2 (3.90 g, 10.0 mmol), stirring in methyl ethyl ketone (25ml), was added ground K₂ CO₃ (2.07 g, 15.0 mmol) followed by1,2-dibromoethane (10 ml). The solution brought to reflux and maintainedat this temperature for 18hr. The mixture was cooled to roomtemperature, filtered, and concentrated. Purification of the cruderesidue by flash column chromatography (8 cm×15 cm silica gel, 50% ethylacetate in hexanes) gave compound 28 as a yellow solid 4.32 g (87%).

¹ H-NMR (CDCl₃) δ7.75-7.78 (d, 2H, J=8.8 Hz), 7.52-7.55 (d, 1H, J=8.9Hz), 7.31-7.35 (m, 3H), 6.94-6.98 (dd,1, J=8.9 Hz, J=2.3 Hz), 6.74-6.78(m, 4H); IR (CHCl₃) 3030, 3015, 2965, 2942, 2835, 1601, 1475, 1253,1240, 1167 cm⁻¹ ; FD⁺ -MS 496(Br79), 498 (Br81); Elemental Analysis C₂₅H₂₁ BrO₄ S - Calculated: C, 60.37; H, 4.26; Br, 16.07; Found: C, 60.22;H, 4.54; Br, 16.20.

Preparation 5

Preparation of compound 29: ##STR16##

Compound 28 (0.5 g, 1.0 mmol) and sodium azide (0.12 g, 2.0 mmol) werestirred in DMF for 144 hours followed by warming to 80° C. for 1 hr. Thesolvent was removed under reduced pressure and the residuechromatographed on silica gel using ethyl acetate/hexanes (1:1),affording 0.41 g (89%) of compound 29.

¹ H-NMR (DMSO-d₆) δ7.64-7.66 (m, 3H), 7.28-7.33 (m, 3H), 6.85-6.99 (m,5H), 4.15-4.18 (m, 2H), 3.82 (s, 3H), 3.65 (s, 3H), 3.60-3.63 (m, 2H);FD⁺ -MS for C₂₅ H₂₁ N₃ O₄ S=459; Elemental Analysis C₂₅ H₂₁ N₃ O₄ S -Calculated: C, 65.35; H, 4.61; N, 9.14; Found: C, 65.55; H, 4.79; N,9.17.

Preparation 6

Preparation of compound 30: ##STR17##

Compound 29 (11.1 g, 24.2 mmol) in 50 ml of THF and 85 ml ethanol, with1.5 g 5% Palladium on carbon was hydrogenated at room temperature for 24hrs. The reaction mixture was filtered, concentrated and recrystallizedfrom ethyl acetate/hexane to afford 6.06 g (58%) of compound 30. The HC₁salt of an aliquot was prepared for physical chemistry characterization.

IR (KBr)ν_(max) 3418, 2937, 2836, 1634, 1598, 1574, 1531, 1498, 1473,1438, 1350, 1294, 1251, 1167, 1112, 1046, 1025, 830; ¹ H-NMR (DMSO-d₆)δ8.22-8.23 (br s, 2H), 7.64-7.61 (t, 3H), 7.28-7.31 (d, 3H), 4.19-4.20(m, 2H), 3.82 (s, 3H), 3.69 (s, 3H), 3.15-3.17 (m, 2H); FD⁺ -MS for C₂₅H₂₄ ClNO₄ S=433; Elemental Analysis C₂₅ H₂₄ ClNO₄ S - Calculated: C,63.89; H, 5.15; N, 2.98; Found: C, 63.80; H, 5.11; N, 2.83.

EXAMPLE 5

Preparation of compound 17: ##STR18##

Compound 30 (1.0 g, 2.3 mmol), benzoyl chloride (0.35 g, 2.5 mmol) andsodium hydroxide (0.1 g, 2.5 mmol) was stirred in 75 ml water at roomtemperature for 18 hrs. The product was extracted with ethyl acetate,dried over sodium sulfate, concentrated and chromatographed on silicagel using an ethyl acetate/methanol gradient yielding 1.03 g (83%) ofcompound 17.

¹ H-NMR (DMSO-d₆) δ8.63 (m, 1H), 7.80-7.82 (m, 1H), 7.63-7.68 (m, 3H),7.40-7.49 (m, 3H), 7.26-7.31 (m, 4H), 6.85-6.98 (m, 5H, 4.19-4.20 (m,2H), 3.15-3.17 (m, 2H); FD⁺ -MS for C₃₂ H₂₇ NO₅ S=537; ElementalAnalysis C₃₂ H₂₇ NO₅ S - Calculated: C, 71.49; H, 5.06; N, 2.60; Found:C, 71.72; H, 5.12; N, 2.62.

The following compounds for which physical data are shown may beprepared in a manner analogous with procedures detailed in the aboveexamples.

EXAMPLE 6

Compound 3: ##STR19##

¹ H NMR (DMSO-d₆) δ9.78(s, 1H), 9.72(s, 1H), 7.66 (d, 2H, J=10 Hz), 7.34(s, 1H), 7.26 (d, 1H, J=10 Hz), 7.18 (d, 2H, J=10 Hz), 6.91 (d, 2H, J=10Hz), 6.84 (dd, 1H, J=10 Hz), 6.66 (d, 2H, J=10 Hz), 4.12 (t, 2H, J=8Hz), 3.58(t, 2H, J=8 Hz), 2.16 (bs, 2H), 1.64 (bs, 4H); EI MS for C₂₈H₂₅ NO₅ S=487 (M*); Elemental Analysis C₂₈ H₂₅ NO₅ S - Calculated: C,68.98; H, 5.17; N, 2.87; Found: C, 69.08; H, 5.08; N, 2.69.

EXAMPLE 7

Compound 4: ##STR20##

¹ H NMR (CDCl₃) δ7.77 (d, 2H, J=10 Hz), 7.53 (d, 1H, J=10 Hz), 7.35 (m,3H), 6.96 (dd, 1H, J=10 Hz, J=3 Hz), 6.75 (dd, 4H, J=10 Hz, J=3 Hz),4.16 (t, 2H, J=8 Hz), 3.89 (s, 3H), 3.74 (s, 3H), 3.68(t, 2H, J=8 Hz),3.45 (bs, 2H), 2.34 (bs, 2H), 1.88 (bs, 4H); FD MS for C₃₀ H₂₉ NO₅ S=515(M*); Elemental Analysis C₃₀ H₂₉ NO₅ S - Calculated: C, 69.88; H, 5.67;N, 2.72; Found: C, 69.71; H, 5.67; N, 2.73.

EXAMPLE 8

Compound 5: ##STR21##

¹ H NMR (CDCl₃) δ7.74 (d, 2H, J=9.7 Hz), 7.53 (d, 1H, J=9.7 Hz), 7.33(m, 3H), 6.97 (dd, 1H, J=9.7 Hz), 6.77 (d, 2H, J=9.7 Hz), 6.73 (d, 2H,J=9.7 Hz), 4.12 (t, 2H, J=9.7 Hz), 3.9 (m, 5H), 2.75(s, 3H), 2.67 (s,4H); FD+MS=515.

EXAMPLE 9

Compound 6: ##STR22##

IR (KBr)ν_(max) 3228, 2973, 1659, 1597, 1537, 1499, 1467, 1420, 1359,1258, 1165, 1116, 1037, 908, 835, 807, 540; ¹ H-NMR (DMSO d₆) δ9.75 (S,1H), 9.71 (s, 1H), 7.56-7.62 (m, 3H), 7.21-7.32 (m, 2H), 6.84-6.91 (d,2H, J=6.3 Hz), 6.66-6.84 (d, 2H), 4.07-4.09 (t, 2H, J=5.3 Hz), 3.47-3.48(t, 2H, J=5.3 Hz), 2.47-2.48 (t, 2H), 2.12-2.18 (m, 2H), 1.84-1.87 (m,2H); FD+MS for C₂₇ H₂₃ NO₅ S=473; Elemental Analysis C₂₇ H₂₃ NO₅ S -Calculated: C, 68.48; H, 4.90; N, 2.96; Found: C, 68.21; H, 5.13; N,2.99.

EXAMPLE 10

Compound 8: ##STR23##

¹ H-NMR (acetone-d₆) δ8.68 (bs, 2H), 7.71 (d, J=8.7 Hz, 2H), 7.33-7.40(m, 2H), 7.26 (d, J=8.9 Hz, 2H), 6.82-6.94 (m, 3H), 6.73 (d, J=8.8 Hz,2H), 4.53 (s, 2H), 3.51 (t, J=4.0 Hz, 2H), 3.36 (t, J=4.1 Hz, 2H),1.71-1.90 (series of m, 4H), IR (CHCl₃) 3307 (b), 1645 cm⁻¹ ; FD⁺ -MS473 (M⁺)

EXAMPLE 11

Compound 9: ##STR24##

¹ H-NMR (acetone-d₆) δ8.68 (bs, 1H), 8.60 (bs, 1H), 7.70 (d, J=8.9 Hz,2H), 7.35-7.41 (m, 2H), 7.27 (d, J=S.9 Hz, 2H), 6.84-6.95 (m, 3H), 6.73(d, J=8.7 Hz, 2H), 4.81 (s, 2H), 3.47 (t, J=4.1 Hz, 4H), 1.43-1.67(series of m, 6H), IR (CHCl₃) 3300 (b), 1639 cm⁻¹ ; FD⁺ -MS 487 (M⁺);Elemental Analysis C₂₈ H₂₅ NO₅ S - Calculated: C, 68.98; H, 5.17; N,2.87; Found: C, 67.50; H, 5.43; N, 2.84.

EXAMPLE 12

Compound 10: ##STR25##

¹ H-NMR (DMSO-d₆) δ9.81 (s, 1H), 9.73 (s, 1H), 7.64 (d, J=9.0 Hz, 2H),7.33 (d, J=2.3 Hz, 1H), 7.15-7.25 (m, 3H), 6.82-6.91 (m, 3H), 6.69 (d,J=8.8 Hz, 2H), 4.85 (s, 2H), 3.20-3.35 (m, 5H), 0.1.5-1.57 (series of m,4H), 0.81-0.92 (m, 3H); IR (CHCl₃) 3300 (b), 1625cm⁻¹ ; FD⁺ -MS 489(M⁺); Elemental Analysis C₂₈ H₂₇ NO₅ S - Calculated: C, 68.69; H, 5.56;N, 2.86; Found: C, 67.75; H, 5.49; N, 2.88.

EXAMPLE 13

Compound 12: ##STR26##

¹ H-NMR (MeOD d₄) δ7.71 (d, 2H, J=9.0 Hz), 7.4 (d, 1H, J=9.0 Hz), 7.26(s, 1H), 7.20 (d, 2H, J=9.0 Hz), 6.87 (m, 2H), 6.42 (d, 2H, J=9.0 Hz),3.05 (s, 3H), 2.96 (s, 3H); FD⁺ -MS=447; Elemental Analysis C₂₅ H₂₁ NO₅S - Calculated: C, 67.10; H, 4.73; N, 3.13; Found: C, 67.32; H, 4.94; N,2.99.

EXAMPLE 14

Compound 13: ##STR27##

¹ H NMR (MeOD d₄) δ7.68 (d, 2H, J=9.0 Hz), 7.41 (d, 1H, J=9.0 Hz), 7.26(s, 1H), 7.18 (d, 2H, J=9.0 Hz), 6.86 (d, 1H, J=9.0 Hz), 6.80 (d, 2H,J=9.0 Hz), 6.63 (d, 2H, J=9.0 Hz), 4.15 (t, 2H, J=6 Hz), 3.84 (t, 2H,J=6 Hz), 2.62 (s, 4H); FD⁺ -MS=487; Elemental Analysis C₂₇ H₂₁ NO₆ S -Calculated: C, 66.52; H, 4.34; N, 2.87; Found: C, 66.65; H, 4.55; N,2.83.

EXAMPLE 15

Compound 14: ##STR28##

¹ H-NMR (acetone-d₆) δ8.63 (bs, 1H) 7.79 (d, J=8.9 Hz, 2H), 7.36-7.46(m, 2H), 7.28 (d, J=9.1 Hz, 2H), 7.02 (d, J=8.9 Hz, 2H), 6.93 (dd, J=8.9Hz, 2.8 Hz, 1H), 6.73 (d, J=9.0, 2H), 5.12 (s, 2H); FD⁺ -MS 401 (M+).

EXAMPLE 16

Compound 15: ##STR29##

¹ H NMR (CDCl₃) δ7.56-7.82 (t, 2H, J=5.8 Hz), 7.33-7.36 (t, 2H, J=2.9Hz), 6.97-6.98 (m, 1H), 6.73-6.80 (m, 4H), 4.08-4.14 (m, 2H), 3.89-3.92(d, 3H, J=8.5 Hz), 3.76-3.78 (d, 3H, J=8.7 Hz), 3.64-3.70 (m, 2H),3.51-3.58 (m, 2H), 2.38-2.43 (m, 2H), 2.03-2.08 (m, 2H); FD⁺ -MS for C₂₉H₂₇ NO₅ S=501.

EXAMPLE 17

Compound 16: ##STR30##

¹ H-NMR δ8.63 (bs, 1H) 7.79 (d, J=8.8 Hz, 2H), 7.38-7.47 (m, 2H), 7.25(d, J=9.0 Hz, 2H), 7.05 (d, J=8.9 Hz, 2H), 6.93 (dd, J=8.7, 2.8 Hz, 1H),6.73 (d, J=9.0,2H), 5.38 (q, J=6.1 Hz, 2H), 1.74 (d, J=6.0 Hz, 3H); FD⁺-MS 415 (M+); Elemental Analysis C₂₄ H₁₇ NO₄ ^(S) - Calculated: C,69.38; H, 4.12; N, 3.37; Found: C 69.19; H, 4.40; N, 3.08.

EXAMPLE 18

Compound 18: ##STR31##

IR (KBr)ν_(max) 3311, 2953, 1637, 1597, 1538, 1502, 1468, 1422, 358,1309, 1258, 1166, 1113, 1038, 907, 836; ¹ H-NMR (DMSO d₆)δ9.68-9.73 (d,2H), 8.63 (m, 1H), 7.76-7.81 (d, 2H, J=8.6 Hz), 7.61-7.64 (d, 2H, J=8.8Hz), 7.38-7.48 (m, 2H), 7.30 (s, 1H), 7.12-7.20 (m, 4H), 6.91-6.93 (m,2H, J=7.0 Hz), 6.81-6.82 (m, 1H), 6.62-6.64 (d, 2H, J=6.6 Hz ),4.11-4.13 (m, 2H), 3.56-3.60 (m, 2H); FD⁺ -MS for C₃₀ H₂₃ NO₅ S=5409;Elemental Analysis C₃₀ H₂₃ NO₅ S - Calculated: C, 70.71; H, 4.55; N,2.75; Found: C, 70.67; H, 4.66; N, 2.68.

EXAMPLE 19

Compound 19: ##STR32##

IR KBr)ν_(max) 3375, 2932, 2856, 1598, 1539, 1506, 1468, 1422, 354,1306, 1258, 1166, 1113, 1035, 907, 836, 647, 620; ¹ H-NMR (DMSO d₆)δ9.70 (br s, 2H), 7.61-7.64 (d, 2H, J=8.4 Hz), 7.50 (s, 1H), 7.21-7.24(d, 1H), 7.13-7.19 (d, 2H), 6.76-6.98 (m, 3H), 6.61-6.63 (d, 2H, J=7.7Hz), 3.40-3.45 (m, 2H), 2.81-2.84 (m, 2H), 0.85-1.81 (m, 11H); FD⁺ -MSfor C₃₁ H₃₁ NO₅ S=529; Elemental Analysis C₃₁ H₃₁ NO₅ S - Calculated:70.30; H, 5.90; N, 2.64; Found: C, 70.43; H, 6.10; N, 2.55.

EXAMPLE 20

Compound 20: ##STR33##

IR (KBr)ν_(max) 3363, 2931, 2854, 1598, 1565, 1503, 1468, 1422, 1357,1315, 1255, 1166, 1038, 908, 836, 808, 672; ¹ H-NMR (DMSO d₆) δ9.70-9.75(d, 2H), 7.62-7.65 (d, 2H, J=7.9 Hz), 7.31 (s, 1H), 7.21-7.24 (d, 1H),7.13-7.16 (d, 2H, J=8.2 Hz), 6.88-6.91 (d, 2H, J=8.3 Hz), 6.81-6.84 (d,1H), 6.64-6.66 (d, 2H, J=8.3 Hz), 3.96-3.99 (m, 2H), 3.30 (m, 2H),1.28-1.60 (m, 5H), 0.96-1.27 (m, 6H); FD⁺ -MS for C₃₀ H₃₀ N₂ O₅ S=530;Elemental Analysis C₃₀ H₃₀ N₂ O₅ S - Calculated: C, 67.91; H, 5.70; N,5.28; Found: C, 68.12; H, 5.99; N, 5.39.

EXAMPLE 21

Compound 21: ##STR34##

¹ H-NMR (DMSO d₆) δ9.70 (s, 2H), 7.60-7.65 (d, 2H, J=8.6 Hz), 7.50 (s,1H), 7.14-7.22 (m, 2H), 6.80-7.00 (m, 4H), 6.62-6.66 (m, 2H), 4.01-4.04(m, 2H), 3.50-3.70 (m, 2H), 1.40-1.78 (m, 5H), 1.01-1.39 (m, 6H); FD⁺-MS for C₃₀ H₂₉ NO₅ S=519.

EXAMPLE 22

Compound 22: ##STR35##

IR (CHCl₃)ν_(max) 3298, 3025, 3010, 2946, 1600, 1262, 1166; ¹ H-NMR(MeOD d₄) δ7.68-7.71 (d, 2H, J=8.8 Hz), 7.38-7.41 (d, 1H, J=8.7 Hz),7.24-7.25 (d, 1H, J=2.-8 Hz), 7.16-7.19 (d, 2H, J=8.6 Hz), 6.83-6.87 (m,3H), 6.60-6.63 (d, 2H, J=8.6 Hz), 4.35-4.20 (m, l1H), 1.25 (s, 9H); ¹³C-NMR (MeOD d₄) δ194.39, 178.33, 163.40, 158.07, 155.62, 142.72, 140.28,133.19, 132.37,130.62, 130.24, 130.09, 124.89, 123.62, 115.38,114.93,114.28, 106.82, 65.17, 50.36, 38.89, 27.70, 25.53, 25.16, 19.09;FD⁺ -MS for C₃₂ H₃₃ O₅ S=543; Elemental Analysis C₃₂ H₃₃ O₅ S -Calculated: C, 70.69; H, 6.12; N, 2.58; Found: C, 70.47; H, 6.13; N,2.34.

EXAMPLE 23

Compound 23: ##STR36##

IR (CHCl₃)ν_(max) 3293, 3021, 3010, 1598, 1254, 1166; ¹ H-NMR (MeOD d₄)δ7.65-7.68 (d, 2H, J=8.9 Hz), 7.45-7.48 (d, 1H, J=8.-8 Hz), 7.25-7.26(d, 1H, J=2.2 Hz), 7.14-7.17 (d, 2H, J=8.6 Hz ), 6.81-6.89 (m, 3H),6.59-6.62 (d, 2H, J=8.6 Hz), 4.63-4.69 (m, 1H), 3.79-3.89 (m, 2H),3.51-3.60 (m, 2H), 1.89-1.95 (m, 2H), 1.62-1.71 (m, 2H), 1.27 (s, 9H);¹³ C-NMR (MeOD d₄) δ194.06, 177.14, 161.67, 157.8, 155.43, 1 43.21,140.04, 132.92, 132.18, 130.38, 130.16, 129.85, 124.71, 123.51, 115.10,115.06, 114.74, 106.57, 71.80, 41.75, 38.40, 3 0.41, 27.28; FD⁺ -MS forC₃₁ H₃₁ O₅ S=529.

EXAMPLE 24

Compound 24: ##STR37##

IR (CHCl₃ )ν_(max) 3008, 1609, 1599, 1476, 1253, 1191, 1188; ¹ H-NMR(CDCl₃) δ7.73-7.76 (d, 2H, J=8.7 Hz), 7.53-7.56 (d, 1H, J=8.9 Hz),7.31-7.34 (m, 3H), 6.94-6.98 (dd, 1H, J=8.-8 Hz, J=2.0 Hz), 6.73-6.76(m, 4H), 4.52-4.60 (m, 1H), 3.88 (s, 3H), 3.74 (s, 3H), 1.67-3.85 (m,8H), 1.28 (s, 9H); ¹³ C-NMR (CDCl₃) δ193.03,176.31, 161.29, 159.73,157.66, 142.78,140.03,133.91, 132.40, 130.58, 130.49, 130.31, 126.03,124.06, 115.14, 114.81, 113.99, 104.46, 71.86, 55.60, 55.23, 41.64,38.67, 30.61, 28.34, 25.57; FD⁺ -MS for C₃₃ H₃₅ O₅ S=557.

EXAMPLE 25

Compound 26: ##STR38##

¹ H-NMR (DMSO d₆) 59.75 (d, 2H), 7.62-7.65 (d, 2H), 7.31 (d, 1H),7.16-7.19 (d, 2H), 6.83-6.91 (m, 4H), 6.66-6.68 (m, 2H), 3.97-3.99 (m,2H), 3.59-3.81 (m, 2H), 1.77 (s, 3H); FD⁺ -MS for C₂₅ H₂₁ NO₅ S=447.

The compounds of formula I, II, or III of the present invention areuseful for alleviating the symptoms of post-menopausal sundrome,particularly osteoporosis, assocated cardiovascular diseases,particularly hyperlipidemia, and estrogen-dependent cancer, particularlyestrogen-dependent breast and uterine carcinoma. The term "alleviating"is defined to include prophylactically treating a person at risk ofincurring one or more symptoms or pathological conditions ofpost-menopauysal syndrome, holding in check such symptoms orpathological conditions, and treating existing symptoms or pathologicalconditions, as appropriate.

Compounds of the present invention are also effective for inhibitinguterine fibroid disease and endometriosis in women, and smooth musclecell proliferation in humans. The following non-limiting biological testexamples illustrate the methods of the present invention.

Biological Test Procedures

I. General Preparation for Post-Menopausal Rat Model

In the examples illustrating the methods, a post-menopausal model wasused in which effects of different treatments upon various biologicalparameters were determined, including serum cholesterol concentration,uterine weight, eosinophil peroxidase activity, MCF-7 cellproliferation, and bone density.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to225 g) were obtained from Charles River Laboratories (Portage, MI). Theanimals were either bilaterally ovariectomized (OVX) or exposed to asham surgical procedure (Intact) at Charles River Laboratories, and thenshipped after one week. Upon arrival, they were housed in metal hangingcages in groups of 3 or 4 per cage and had ad libitum access to food(calcium content approximately 0.5%) and water for one week. Roomtemperature was maintained at 22.2° ±1.7° C. with a minimum relativehumidity of 40%. The photoperiod in the room was 12 hours light and 12hours dark.

II. Four Day D©Sing Regimen

After a one week acclimation period (therefore, two weeks post-OVX),daily dosing with test compound was initiated. 17α-Ethynyl estradiol(EE₂) (Sigma Chemical Co., St. Louis, Mo.), an orally available form ofestrogen, or the test compound were given orally, unless otherwisestated, as a suspension in 1% carboxymethyl cellulose or dissolved in20% cyclodextrin. Animals were dosed daily for 4 days. Following thedosing regimen, animals were weighed and anesthetized with aketamine:xylazine (2:1, v:v) mixture. A blood sample was collected bycardiac puncture. The animals were then sacrificed by asphyxiation withCO₂, the uterus was removed through a midline incision, and a wetuterine weight was determined.

A. Cholesterol Analysis

Blood samples were allowed to clot at room temperature for 2 hours, andserum was obtained following centrifugation for 10 minutes at 3000 rpm.Serum cholesterol was determined using a Boehringer Mannhelm Diagnosticshigh performance cholesterol assay. Briefly the cholesterol was oxidizedto cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide wasthen reacted with phenol and 4-aminophenazone in the presence ofperoxidase to produce a p-quinone imine dye, which was readspectrophotemetrically at 500 nm. Cholesterol concentration was thencalculated against a standard curve. The entire assay was automatedusing a Biomek Automated Workstation.

B. Uterine Eosinophil Peroxidase (EPO) Assay

Uteri were kept at 4° C. until time of enzymatic analysis. The uteriwere then homogenized in 50 volumes of 50 mM Tris buffer (pH - 8.0)containing 0.005% Triton X-100. Upon addition of 0.01% hydrogen peroxideand 10 mM o-phenylenediamine (final concentrations) in Tris buffer,increase in absorbance was monitored for one minute at 450 nm. Thepresence of eosinophils in the uterus, as measured by assay ofeosinophil peroxidase activity, is an indication of estrogenic activityof a compound. The maximal velocity of a 15 second interval wasdetermined over the initial, linear portion of the reaction curve.

C. Results

Data presented in Table 1 below show comparative results among controlovariectomized rats, rats treated with EE2, and rats treated withcertain compounds of the present invention. Although EE2 caused adecrease in serum cholesterol when orally administered at 0.1 mg/Kg/day,it also exerted a marked stimulatory action on the uterus so that theuterine weight of EE2 treated rats was substantially greater than theuterine weight of ovariectomized test animals. This uterine response toestrogen is well recognized in the art.

In contrast, the compounds of the present invention substantially reduceserum cholesterol compared to the ovariectomized control animals withoutthe general increase of uterine weight that is associated with estrogencompounds known in the art. This benefit of serum cholesterol reductionwithout adversely affecting uterine weight is quite rare and desirable.

As is expressed in the data below, estrogenicity also was assessed byevaluating the adverse response of eosinophil infiltration into theuterus. The compounds of the present invention did not cause an increasein the number of eosinophils observed in the stromal layer ofovariectomized rats, or in rare instances an increase only at thehighest concentrations tested, as measured by assay of eosinophilperoxidase activity, while EE₂ caused a substantial, expected increasein eosinophil infiltration.

The data presented in Table 1 reflect the response of 5 to 6 rats pertreatment.

                  TABLE 1                                                         ______________________________________                                                         Uterine    Uterine                                                                              Serum                                              Dose     Weight (%  EPO    Cholesterol                                Compound                                                                              (mg/kg)  inc. OVX)  (Vmax) (% dec. OVX)                               ______________________________________                                        ethynyl 0.1      235.2*     120.0* 89.6*                                      estradiol                                                                      3      0.1      13.3       8.0    38.1*                                              1        17.8       7.8    45.8*                                              10       57.6*      8.9    72.4*                                       6      0.1      -9         3.6    33.6*                                              1        15.4       1.4    52.4*                                              10       40.3*      3.7    67.5*                                       7      0.1      -0.5       5.2    3.4                                                1        -16.2      3.7    14.6                                               10       8.4        4.7    -41.9*                                      8      0.1      -4.7       3.6    17.4                                               1        25.6*      6.1    69.6*                                              10       61.7*      39.1   70.3*                                       8      0.01     -22.9      3.5    27.2                                               0.1      -12.6      4.4    25.8                                               1        -11.5      3.7    39.4*                                              10       -1.3       3.7    64.5*                                       9      0.1      -10.0      2.8    4.2                                                1        6.5        3.6    22.0                                               10       51.7*      32     74.3*                                      10      0.1      1.5        4.3    -40.0*                                             1        -6.7       3.1    19.1                                               10       38.0*      17.0   67.1*                                      12      0.1      -7.7       3.7    27.8*                                              1        15.4       6.7    48.2*                                              10       60.7*      122.3* 71.2*                                      13      0.1      22.2       1.9    37.6*                                              1        7.2        1.7    12.6                                               10       -4.0       2.5    30.1*                                      14      0.1      18.8       3.3    3.3                                                1        14.6       2.4    23.7                                               10       124.6*     145.8* 91.1                                       16      0.1      0.7        2.3    3.4                                                1        27.4       3.9    28.8*                                              10       99.2*      23.4   71.2*                                      18      0.1      16.1       3.2    -10.8                                              1        25.7       3.6    -12.2                                              10       12.6       3.6    12.8                                       19      0.1      7          5.4    14.5                                               1        -25.5*     3.7    20.1                                               10       47.0*      29.3*  52.2*                                      20      0.1      -29.9      3.0    -27.2                                              1        -27.4      2.5    -6.0                                               10       -32.7      2.9    -15.4                                      21      0.1      -5.5       3.1    9.3                                                1        -11.5      2.6    0.5                                                10       8.6        2.4    24.0*                                      22      0.1      1.9        3.5    22.1*                                              1        19.6       20.0   36.2*                                              10       46.9*      69.5*  71.5*                                      23      0.1      -4.3       1.6    -17.4                                              1        0.8        3.8    -5.2                                               10       22.3       6.7    55.8*                                      26      0.1      -17.5      1.3    15.8                                               1        -28.8*     0.7    19.0                                               10       25.8*      13.6   32.8                                       ______________________________________                                         *indicates value is significantly different than OVX control.            

In addition to the demonstrated benefits of the compounds of the presentinvention, especially when compared to estradiol, the above data clearlydemonstrate that these compounds are not estrogen mimetics. Furthermore,no deleterious toxicological effects (survival) were observed withtreatment by any of the compounds of the present invention.

Ill. Thirty-Five Day Dosing Regimen

Following the General Preparation procedure described above, the ratswere treated daily for 35 days (6 rats per treatment group) andsacrificed by decapitation on the 36th day. The 35 day time period wassufficient to allow maximal effect on bone density, measured asdescribed herein. At the time of sacrifice, the uteri were removed,dissected free of extraneous tissue, and the fluid contents wereexpelled before determination of wet weight in order to confirm estrogendeficiency associated with complete ovariectomy. Uterine weight wasroutinely reduced about 75% in response to ovariectomy. The uteri werethen placed in 10% neutral buffered formalin to allow for subsequenthistological analysis.

A. Bone Density Assay

The right tibias were excised and scanned at the distal metaphysis 1 mmfrom the patellar groove with single photon absorptiometry. Results ofthe densitometer measurements represent a calculation of bone density asa function of the bone mineral content and bone width.

In accordance with the procedures outlined above, compounds of thepresent invention and EE₂ in 20% hydroxypropyl β-cyclodextrin wereorally administered to test animals.

B. Results

Ovariectomy of the test animals caused a significant reduction in tibiadensity compared to intact, vehicle treated controls. Orallyadministered EE2 prevented this loss, but the risk of uterinestimulation with this treatment is ever-present.

The compounds of the present invention also prevented bone loss in ageneral, dose-dependent manner. Accordingly, the compounds of thepresent invention are useful for the treatment of post-menopausalsyndrome, particularly osteoporosis.

IV. MCF-7 Proliferation Assay

HCF-7 breast adenocarcinoma cells (ATCC HTB 22) were maintained in MEM(minimal essential medium, phenol red-free, Sigma, St. Louis, MO)supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2mM), sodium pyruvate (1 mM), HEPES((N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]10 mM),non-essential amino acids and bovine insulin (1 μg/ml) (maintenancemedium). Ten days prior to assay, MCF-7 cells were switched tomaintenance medium supplemented with 10% dextrancoated charcoal strippedfetal bovine serum (DCC-FBS) assay medium) in place of 10% FBS todeplete internal stores of steroids. MCF-7 cells were removed frommaintenance flasks using cell dissociation medium (Ca++/Mg++ free HBSS(phenol red-free) supplemented with 10 mM HEPES and 2 mM EDTA). Cellswere washed twice with assay medium and adjusted to 80,000 cells/mi.Approximately 100 ml (8,000 cells) were added to flat-bottommicroculture wells (Costar 3596) and incubated at 37° C. in a 5% CO₂humidified incubator for 48 hours to allow for cell adherence andequilibration after transfer. Serial dilutions of drugs or DMSO as adiluent control were prepared in assay medium and 50 ml transferred totriplicate microcultures followed by 50 ml assay medium for a finalvolume of 200 ml. After an additional 48 hours at 37° C. in a 5% CO₂humidified incubator, microcultures were pulsed with tritiated thymidine(1 uCi/well) for 4 hours. Cultures were terminated by freezing at -70°C. for 24 hours followed by thawing and harvesting of microcultures.Samples were counted by liquid scintillation. Results in Table 3 showthe ED₅₀ for certain compounds of the present invention.

                  TABLE 3                                                         ______________________________________                                                       ED.sub.50                                                             Compound                                                                              (nM)                                                           ______________________________________                                               3       600                                                                   7       >1000                                                                 8       10                                                                    9       10                                                                    10      50                                                                    13      >1000                                                                 14      0.05                                                                  16      0.02                                                                  18      500                                                                   19      0.1                                                                   20      0.1                                                                   22      0.01                                                                  23      4.0                                                            ______________________________________                                    

V. DMBA-Induced Mammary Tumor Inhibition

Estrogen-dependent mammary tumors are produced in female Sprague-Dawleyrats which are purchased from Harlan Industries, Indianapolis, Indiana.At about 55 days of age, the rats receive a single oral feeding of 20 mgof 7,12-dimethylbenz[a]anthracene (DMBA). About 6 weeks after DMBAadministration, the mammary glands are palpared at weekly intervals forthe appearance of tumors. Whenever one or more tumors appear, thelongest and shortest diameters of each tumor are measured with a metriccaliper, the measurements are recorded, and that animal is selected forexperimentation. An attempt is made to uniformly distribute the varioussizes of tumors in the treated and control groups such thataverage-sized tumors are equivalently distributed between test groups.Control groups and test groups for each experiment contain 5 to 9animals.

Compounds of the current invention are administered either throughintraperitoneal injections in 2% acacia, or orally. Orally administeredcompounds are either dissolved or suspended in 0.2 ml corn oil. Eachtreatment, including acacia and corn oil control treatments, isadministered once daily to each test animal. Following the initial tumormeasurement and selection of test animals, tumors are measured each weekby the above-mentioned method. The treatment and measurements of animalscontinue for 3 to 5 weeks at which time the final areas of the tumorsare determined. For each compound and control treatment, the change inthe mean tumor area is determined.

VI. Uterine Fibrosis Test Procedures

Test 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Test 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4

A. Induction of fibroid tumors in guinea pig

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Uterine fibroid tissue implantation in nude mice

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, female, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the time of sacrifice, the uteri is harvested to assessthe status of the organ.

Test 5

Tissue from human uterine fibroid tumors is harvested and maintained, invitro, as primary nontransformed cultures. Surgical specimens are pushedthrough a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the present invention and vehicle.Levels of steroid hormone receptors are assessed weekly to determinewhether important cell characteristics are maintained in vitro. Tissuefrom 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

VII. Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Test 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections of water for 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Test 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Test 3

A. Surgical induction of endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level of hormone. Autologousendometrial tissue is implanted in the peritoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting of a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Endometrial tissue implantation nude mice

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Test 4

Tissue from human endometrial lesions is harvested and maintained invitro as primary nontransformed cultures. Surgical specimens are pushedthrough a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the invention, and vehicle. Levels ofsteroid hormone receptors are assessed weekly to determine whetherimportant cell characteristics are maintained in vitro. Tissue from 5-25patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

VIII. Inhibition of Aortal Smooth Cell Proliferation/Restenosis TestProcedure

Compounds of the present invention have capacity to inhibit aortalsmooth cell proliferation. This can be demonstrated by using culturedsmooth cells derived from rabbit aorta, proliferation being determinedby the measurement of DNA synthesis. Cells are obtained by explantmethod as described in Ross, J. of Cell Bio. 50:172 (1971). Cells areplated in 96 well microtiter plates for five days. The cultures becomeconfluent and growth arrested. The cells are then transferred toDulbecco's Modified Eagle's Medium (DMEM) containing 0.5-2% plateletpoor plasma, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/mlstreptomycin, 1 mC/ml ³ H-thymidine, 20 ng/ml platelet-derived growthfactor, and varying concentrations of the present compounds. Stocksolution of the compounds is prepared in dimethyl sulphoxide and thendiluted to appropriate concentration (0.01-30 mM) in the above assaymedium. Cells are then incubated at 37° C. for 24 hours under 5% CO₂/95% air. At the end of 24 hours, the cells are fixed in methanol. ³ Hthymidine incorporation in DNA is then determined by scintillationcounting as described in Bonin, et al., Exp. Cell Res. 181: 475-482(1989).

Inhibition of aortal smooth muscle cell proliferation by the compoundsof the present invention are further demonstrated by determining theireffects on exponentially growing cells. Smooth muscle cells from rabbitaortae are seeded in 12 well tissue culture plates in DMEM containing10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin. After 24 hours, the cells are attached and themedium is replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100U/ml penicillin, 100 μg/ml streptomycin, and desired concentrations ofthe compounds. Cells are allowed to grow for four days. Cells aretreated with trypsin and the number of cells in each culture isdetermined by counting using a ZM-Coulter counter.

Activity in the above tests indicates that the compounds of the presentinvention are of potential in the treatment of restenosis.

Combination Therapy

The present invention also provides a method of alleviatingpost-menopausal syndrome in women which comprises the aforementionedmethod using compounds of the present invention and further comprisesadministering to a woman an effective amount of estrogen or progestin.These treatments are particularly useful for treating osteoporosis andlowering serum cholesterol because the patient will receive the benefitsof each pharmaceutical agent while the compounds of the presentinvention would inhibit undesirable side-effects of estrogen andprogestin. Activity of these combination treatments in any of thepost-menopausal tests, supra, indicates that the combination treatmentsare useful for alleviating the symptoms of post-menopausal symptoms inwomen.

Various forms of estrogen and progestin are commercially available.Estrogen-based agents include, for example, ethenyl estrogen (0.01-0.03mg/day), mestranol (0.05-0.15 mg/day), and conjugated estrogenichormones such as Premarin® (Wyeth-Ayerst; 0.3-2.5 mg/day).Progestin-based agents include, for example, medroxyprogesterone such asProvera® (Upjohn; 2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), andnonethindrone (0.5-2.0 mg/day). A preferred estrogen-based compound isPremarin, and norethylnodrel and norethindrone are preferredprogestin-based agents.

The method of administration of each estrogen- and progestin-based agentis consistent with that which is known in the art. For the majority ofthe methods of the present invention, compounds of the present inventionare administered continuously, from 1 to 3 times daily. However,cyclical therapy may especially be useful in the treatment ofendometriosis or may be used acutely during painful attacks of thedisease. In the case of restenosis, therapy may be limited to short (1-6months) intervals following medical procedures such as angioplasty.

As used herein, the term "effective amount" means an amount of compoundof the present invention which is capable of alleviating the symptoms ofthe various pathological conditions herein described. The specific doseof a compound administered according to this invention will, of course,be determined by the particular circumstances surrounding the caseincluding, for example, the compound administered, the route ofadministration, the state of being of the patient, and the pathologicalcondition being treated. A typical daily dose will contain a nontoxicdosage level of from about 5 mg to about 600 mg/day of a compound of thepresent invention. Preferred daily doses generally will be from about 15mg to about 80 mg/day.

The compounds of this invention can be administered by a variety ofroutes including oral, rectal, transdermal, subucutaneus, intravenous,intramuscular, and intranasal. These compounds preferably are formulatedprior to administration, the selection of which will be decided by theattending physician. Thus, another aspect of the present invention is apharmaceutical composition comprising an effective amount of a compoundof the current invention, optionally containing an effective amount ofestrogen or progestin, and a pharmaceutically acceptable carrier,diluent, or excipient.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceutically acceptable" itis meant the carrier, diluent, excipients, and salt must be compatiblewith the other ingredients of the formulation, and not deleterious tothe recipient thereof.

Pharmaceutical formulations of the present invention can be prepared byprocedures known in the art using well known and readily availableingredients. For example, the compounds of the current invention, withor without an estrogen or progestin compound, can be formulated withcommon excipients, diluents, or carriers, and formed into tablets,capsules, suspensions, powders, and the like. Examples of excipients,diluents, and carriers that are suitable for such formulations includethe following: fillers and extenders such as starch, sugars, mannitol,and silicic derivatives; binding agents such as carboxymethyl celluloseand other cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carrierssuch as kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyethyl glycols.

The compounds also can be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for example, by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular physiological location,possibly over a period of time. The coatings, envelopes, and protectivematrices may be made, for example, from polymeric substances or waxes.

Compounds of the present invention, alone or in combination with apharmaceutical agent of the present invention, generally will beadministered in a convenient formulation. The following formulationexamples only are illustrative and are not intended to limit the scopeof the present invention.

Formulations

In the formulations which follow, "active ingredient" means a compoundof formula I, II, or III.

Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Active ingredient 0.1-1000                                                    Starch, NF        0-650                                                       Starch flowable powder                                                                          0-650                                                       Silicone fluid 350 centistokes                                                                  0-15                                                        ______________________________________                                    

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2: Tablets

    ______________________________________                                        Ingredient       Quantity (mg/tablet)                                         ______________________________________                                        Active ingredient                                                                               2.5-1000                                                    Cellulose, microcrystalline                                                                    200-650                                                      Silicon dioxide, fumed                                                                         10-650                                                       Stearate acid    5-15                                                         ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of active ingredientare made up as follows:

Formulation 3: Tablets

    ______________________________________                                        Ingredient          Quantity (mg/tablet)                                      ______________________________________                                        Active ingredient   25-1000                                                   Starch              45                                                        Cellulose, microcrystalline                                                                       35                                                        Polyvinylpyrrolidone                                                                              4                                                         (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                    4.5                                                       Magnesium stearate  0.5                                                       Talc                1                                                         ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

Formulation 4: Suspensions

    ______________________________________                                        Ingredient             Quantity (mg/5 ml)                                     ______________________________________                                        Active ingredient      0.1-1000 mg                                            Sodium carboxymethyl cellulose                                                                       50       mg                                            Syrup                  1.25     mg                                            Benzoic acid solution  0.10     ml                                            Flavor                 q.v.                                                   Color                  q.v.                                                   Purified water to      5        ml                                            ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 5: Aerosol

    ______________________________________                                        Ingredient           Quantity (% by weight)                                   ______________________________________                                        Active ingredient    0.25                                                     Ethanol              25.75                                                    Propellant 22 (Chlorodifluoromethane)                                                              70.00                                                    ______________________________________                                    

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

Formulation 6: Suppositories

    ______________________________________                                        Ingredient         Quantity (mg/suppository)                                  ______________________________________                                        Active ingredient  250                                                        Saturated fatty acid glycerides                                                                  2,000                                                      ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

Formulation 7: Intravenous Solution

    ______________________________________                                        Ingredient             Quantity                                               ______________________________________                                        Active ingredient      50     mg                                              Isotonic saline        1,000  ml                                              ______________________________________                                    

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 ml per minute.

Formulation 8: Combination Capsule I

    ______________________________________                                        Ingredient     Quantity (mg/capsule)                                          ______________________________________                                        Active ingredient                                                                            50                                                             Premarin       1                                                              Avicel pH 101  50                                                             Starch 1500    117.50                                                         Silicon Oil    2                                                              Tween 80       0.50                                                           Cab-O-Sil      0.25                                                           ______________________________________                                    

Formulation 9: Combination Capsule II

    ______________________________________                                        Ingredient     Quantity (mg/capsule)                                          ______________________________________                                        Active ingredient                                                                            50                                                             Norethylnodrel 5                                                              Avicel pH 101  82.50                                                          Starch 1500    90                                                             Silicon Oil    2                                                              Tween 80       0.50                                                           ______________________________________                                    

Formulation 10: Combination Tablet

    ______________________________________                                        Ingredient     Quantity (mg/capsule)                                          ______________________________________                                        Active ingredient                                                                            50                                                             Premarin       1                                                              Corn Starch NF 50                                                             Povidone, K29-32                                                                             6                                                              Avicel pH 101  41.50                                                          Avicel pH 102  136.50                                                         Crospovidone XL10                                                                            2.50                                                           Magnesium Stearate                                                                           0.50                                                           Cab-O-Sil      0.50                                                           ______________________________________                                    

We claim:
 1. A compound with nitrogen-containing non-basic side chainsof formula II ##STR39## wherein R¹ and R², independently, are H, OH,O(C₁ -C₆ alkyl), O--C(O)--(C₁ -C₆ alkyl), O--C(O)--O(C₁ -C₆ alkyl),O--C(O)--Ar, O--C(O)--O--Ar, O--SO₂ -(C₄ -C₆ alkyl), chloro, fluoro, orbromo;W is CHOH, C(O), or CH₂ ; Y is (CH₂) n, or CH (C₁ -C₄ alkyl); V isS, O, or CH₂ CH₂ ; n is 1, 2, or 3; and Ar is optionally substitutedphenyl.
 2. The compound of claim 1 wherein V is S.
 3. The compound ofclaim 1 wherein W is C(O).
 4. The compound of claim 1 wherein Y is CH₂or CHCH₃.
 5. The compound of claim 1 wherein R¹ and R², independently,are OH or O(C₁ -C₆ alkyl).
 6. The compound of claim 1 wherein V is S,and W is C(O), and Y is CH₂ or CHCH₃.
 7. The compound of claim 6 whereinR¹ and R², independently, are OH or OCH₃.
 8. The compound of claim 7wherein Y is CH₂.
 9. The compound of claim 7 wherein Y is CHCH₃.
 10. Apharmaceutical composition comprising a compound according to claim 1,and optionally an effective amount of estrogen or progestin incombination with a pharmaceutically acceptable carrier, diluent, orexcipient.
 11. A pharmaceutical composition comprising a compoundaccording to claim 6, and optionally an effective amount of estrogen orprogestin in combination with a pharmaceutically acceptable carrier,diluent, or excipient.
 12. A pharmaceutical composition comprising acompound according to claim 7, and optionally an effective amount ofestrogen or progestin in combination with a pharmaceutically acceptablecarrier, diluent, or excipient.
 13. A pharmaceutical compositioncomprising a compound according to claim 8, and optionally an effectiveamount of estrogen or progestin in combination with a pharmaceuticallyacceptable carrier, diluent, or excipient.
 14. A pharmaceuticalcomposition comprising a compound according to claim 9, and optionallyan effective amount of estrogen or progestin in combination with apharmaceutically acceptable carrier, diluent, or excipient.